ZFIN ID: ZDB-PUB-040609-13
Novel Vector Systems Optimized for Injecting in vitro-synthesized mRNA into Zebrafish Embryos
Ro, H., Soun, K., Kim, E.J., and Rhee, M.
Date: 2004
Source: Molecules and cells   17(2): 373-376 (Journal)
Registered Authors: Ro, Hyunju
Keywords: none
MeSH Terms:
  • Animals
  • Genes, Reporter
  • Genetic Vectors*
  • In Situ Hybridization
  • Microinjections
  • RNA, Messenger/metabolism*
  • Transfection/methods*
  • Zebrafish/anatomy & histology
  • Zebrafish/embryology*
  • Zebrafish/genetics
PubMed: 15179057
Microinjection of nucleic acids or proteins is a useful way of studying embryonic development. In particular, injection of in vitro-transcribed capped RNA is commonly employed to achieve ectopic or increased expression of genes. Two vector systems, pCS2+ and pT7Ts, have been used for this purpose in zebrafish. However, they were initially optimized for Xenopus embryos not for zebrafish. Here we describe a vector, pcGlobin2, optimized for zebrafish, and its derivative, pcGlobin2-GST. This new vector system offers several advantages. First, pcGlobin 2 contains three critical elements [5 cent and 3 cent zebrafish b-globin UTRs, and a poly(A) tail] for generating stable mRNAs and greatly improving translation efficiency. Second, subcloning and preparation of template DNA is easier because of the larger number of restriction sites. Third, protein-binding assays can be performed directly on the injected embryos with pcGlobin2-GST. Lastly, this vector system can be transfected into animal cells without additional subcloning.